Overexpression of recombinant proteins in host systems is a very common tool in modern biotechnology. Following expression the recombinant proteins are detected, isolated and purified. Recently, purification and detection of the proteins has been simplified by the use of fusion tags. Commonly used fusion tags include the polyhistidine tag and GST tag (Smith et al., (1988) Gene 67:31-40; U.S. Pat. Nos. 5,284,933; and 5,310,663). An advantage of polyhistidine tags is that they can readily be coordinated with divalent metal cations such as nickel, cobalt, copper and zinc and the divalent cations can then be coordinated with a chelating agent which in turn can be coupled to a suitable probe or matrix. (US 2005/0123932 A1; U.S. Pat. No. 5,674,677; U.S. Pat. No. 4,569,794, U.S. Pat. No. 5,047,513 and U.S. Pat. No. 6,479,300).
A variety of biological applications such as in vitro and in vivo diagnostic assays and in vivo therapies require methods of detecting, purifying and transporting biomolecules such as proteins, peptides, oligonucleotides, carbohydrates, etc. A number of techniques have been developed in the past to address these issues (Greg T. Hermanson, Bioconjugate Techniques, Academic Press). Traditional detection techniques utilize radioactive isotopes or fluorescent compounds to monitor probe-target interactions.
Enzyme linked immunosorbent assays (ELISAs) are widely used methods for diagnosing a wide variety of disease states in man and animals, especially those diseases which are characterized by the presence of specific antibodies in the serum. Immunoblot assays are also commonly used to detect specific proteins to analyze the level of expression and the integrity of a protein of interest. Another common application is in the Western blot technique where a protein lysate sample is separated on an SDS gel and the protein bands transferred onto a membrane. The membrane is incubated with a primary antibody that binds the protein of interest, and the the protein is detected using a secondary antibody conjugate that binds to the primary antibody. The secondary antibody is usually detectably labeled with an enzyme, but can also be labeled with probes such as fluorophores. Conjugating chelators to biomolecules is a relatively new technique that can be utilized in immunoassays. In U.S. Pat. No. 5,840,834, the inventors disclose joining two amino acid sequences with an electron acceptor moiety and a linking moiety.
Enzyme chelator conjugates, for example an HRP-NTA conjugate, have been prepared and been used for the detection of polyhistidine tagged proteins (U.S. Pat. No. 5,674,677). However, the conjugation method described is limited to enzymes that contain carbohydrate moieties that may be oxidized to produce reactive aldehyde groups. The NTA group is coupled to the enzyme via a Schiff base which is unstable unless reduced. The disadvantages of this technique are: (1) the lower efficiency of the conjugation reaction, (2) the need to reduce the Schiff base, which can compromise the activity of the enzyme and (3) requirement that the enzyme be a glycoproteins thereby limiting the types of biomolecules that can be modified.
The covalent attachment of chelators to biomolecules also offers a convenient tool to transport transition metal binding proteins/peptides to a target. An antibody bound to a polyhistidine sequence labeled with divalent metal ions such as nickel can be transported to specific cell receptors. Antibodies labeled with different chelators have been employed to transport radio-isotopes to a specific target for therapeutic purposes (U.S. Pat. No. 4,741,900, U.S. Pat. No. 5,756,685, US 2004/0156780).
A large number of procedures have traditionally been used to purify proteins, such as various chromatographic separations (by size, charge or affinity). Proteins that have been genetically modified to have a run of histidines (a histidine tag) can be purified on a nickel column (Hochuli et al. (1987), J. Chromat. 411:177-184; Porath (1992), Protein Exp. Purif. 2:263-281),and a number of commercial vendors provide systems for overexpressing and purifying such proteins. These tagged proteins can also be attached to substrates for manipulation, if desired.
There is no current method of coupling a chelator to a biomolecule which overcomes the disadvantages of the methods discussed above. There exists a need to simplify the procedure of detection, purification and transport of biomolecules such as proteins. The absence of a general method puts a substantial constraint on the usefulness of many molecules. It is apparent, therefore, that new methods for detecting, transporting and purifying biomolecules are greatly to be desired.